Contrast-enhanced microCT (EPIC-μCT) ex vivo applied to the mouse and human jaw joint

Abstract

Objectives: The temporomandibular joint (TMJ) is susceptive to the development of osteoarthritis (OA). More detailed knowledge of its development is essential to improve our insight into TMJ-OA. It is imperative to have a standardized reliable three-dimensional (3D) imaging method that allows for detailed assessment of both bone and cartilage in healthy and diseased joints. We aimed to determine the applicability of a contrast-enhanced microCT (µCT) technique for ex vivo research of mouse and human TMJs.

Methods: Equilibrium partitioning of an ionic contrast agent via µCT (EPIC-µCT) was previously applied for cartilage assessment in the knee joint. The method was ex vivo, applied to the mouse TMJ and adapted for the human TMJ.

Results: EPIC-µCT (30-min immersion time) was applied to mouse mandibular condyles, and 3D imaging revealed an average cartilage thickness of 110 ± 16 µm. These measurements via EPIC-µCT were similar to the histomorphometric measures (113 ± 19 µm). For human healthy OA-affected TMJ samples, the protocol was adjusted to an immersion time of 1 h. 3D imaging revealed a significant thicker cartilage layer in joints with early signs of OA compared with healthy joints (414.2 ± 122.6 and 239.7 ± 50.5 µm, respectively). A subsequent significant thinner layer was found in human joints with late signs of OA (197.4 ± 159.7 µm).

Conclusions: The EPIC-µCT technique is effective for the ex vivo assessment of 3D cartilage morphology in the mouse as well as human TMJ and allows bone-cartilage interaction research in TMJ-OA.

Keywords: Hexabrix; articular cartilage; osteoarthritis; temporomandibular joint.

Figure 1

(a, b) MicroCT (µCT) sagittal cross-section of a condylar sample before and after contrast enhancement. The articular cartilage (AC) showed higher attenuation owing to the immersion of the contrast medium and allowed segmentation. (c) Selection of the regions of interest (scanning-medium = black; articular cartilage = grey; and subchondral bone plate = white) and (d) an example of the produced attenuation-histogram with the relative occurrence of the corresponding voxels. EPIC-µCT, equilibrium partitioning of an ionic contrast agent via µCT.

Figure 2

(a) A strong linear relationship between measurements of cartilage thickness obtained by equilibrium partitioning of an ionic contrast agent via microCT (EPIC-µCT) and histology. (b) The differences (histology—EPIC-µCT) vs average cartilage thickness measured by histology and EPIC-µCT with 95% limits of agreement. N = 12 comparisons between histomorphometric measurement and the corresponding µCT section. SD, standard deviation.

Figure 3

Equilibrium partitioning of an ionic contrast agent via microCT (EPIC-µCT) technique applied ex vivo to the mouse jaw joint. (a, b) In the µCT image, a frontal cross-section of the left jaw joint is shown and its three-dimensional reconstruction produced with conventional µCT. (c) With the EPIC-µCT technique, the visibility of the articular cartilage is enhanced. A dual-threshold procedure allowed segmentation of the cartilage. (d) The cartilage thickness measured with EPIC-µCT was compared with histological sections (Alcian blue staining). (e) A thickness map of the cartilage layer (inferior and sagittal view) could be generated with a direct distance transformation algorithm and presented as a pseudocolour scaled image (i.e. blue to red; increasing cartilage thickness).

Figure 4

The average cartilage attenuation in the three groups as function of immersion time (n = 5 samples per group). A significant increase in cartilage attenuation was seen from 0 to 30 min. After 60 min, there were no significant changes seen in any of the groups (values are mean ± standard deviation). OA, osteoarthritis.

Figure 5

Equilibrium partitioning of an ionic contrast agent via microCT (EPIC-µCT) technique applied ex vivo to the human jaw joint. (a) In the µCT image, a sagittal cross-section of a right human jaw joint and its three-dimensional (3D) reconstruction produced with conventional µCT are shown. (b) With the EPIC-µCT technique, the visibility of the articular cartilage is enhanced. (c) A dual-threshold procedure allows for 3D reconstruction of both bone tissue (transparent structure) and overlying cartilage layer (purple structure). (d) A thickness map of the cartilage layer (inferior view; 150 slices width) is presented as a pseudocolour-scaled image (i.e. blue to red; increasing cartilage thickness).

Figure 6

Three-dimensional articular thickness measurements were performed for the three groups of human samples. Differences were found between the average thicknesses of the cartilage layers between the groups. Furthermore, local differences in cartilage thickness were evident when the groups were compared. OA, osteoarthritis.